Objectives: Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related mortality in the United States, and obesity is a known risk factor for PDAC. Higher circulating levels of fatty acid binding protein 4 (FABP4) is noted in patients with severe obesity and linked to the progression of obesity-related cancers. We have already identified the HTS01037 (HTS), an inhibitor of FABP4. Based on these findings, we evaluated the effects of FABP4 and HTS in PDAC progression in vitro and in vivo.

Methods: KPC cells, murine PDAC cell line with KRASG12D and p53 mutation isolated from genetic PDAC model with KRASG12D and p53 mutation (KPC mice) with C57BL/6J background, were used in all experiments. The effects of FABP4 and HTS on cellular proliferation rate was measured using the MTS assay. Cell cycle analysis and apoptosis analysis were analyzed by flow cytometer. Whole cell ROS was quantified by Amplex Red assay. To evaluate the effect of FABP4 on the syngeneic tumor model, KPC cells were injected subcutaneously into the flank of whole body FABP4 null (AKO) mice and FABP4 +/- heterozygous (WT) mice. Tumor volume was measured three times weekly. Furthermore, we also assess the effect of HTS by using the C57BL/6J mice injected KPC cells subcutaneously into the flank. Two weeks after KPC cells inoculation, treatment was started. HTS 5 mg/kg group of mice received HTS and control group of mice received vehicle alone at day 0 by intraperitoneal administration. At the end of experiments, mice were sacrificed. The tumor volume was compared to control group.

Results: FABP4 increased KPC cell proliferation and significantly facilitated the G1 phase cells to entry into the S and G2 phases (release from G1 arrest). HTS inhibited the activation of cell cycle induced by FABP4. HTS significantly suppressed KPC cell proliferation and inhibited the activation of cell cycle induced by FABP4. Furthermore, the percentages of late-stage apoptotic cells were increased by the HTS. ROS decreased along with exogenous FABP4 treatment. In reverse, HTS significantly increased ROS compared with FABP4. The FABP4 knockout significantly inhibited the tumor growth, and the average tumor volumes of the AKO mice and WT mice were 744.0 ± 273.2 and 1186.7 ± 328.1 mm3, respectively at day 28 after the KPC cells inoculation (Figure A). HTS also decreased the KPC tumor growth and tumors treated with HTS significantly showed higher number of apoptotic cells assessed by TUNEL assay compared to control (Figure B).

Conclusion: FABP4 promoted the PDAC progression and FABP4 inhibition showed significant antitumor effect by suppressing proliferation in mouse syngeneic tumor model. FABP4 can be a critical therapeutic target for obesity-related PDAC.

Masato Yamamoto

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